ABSTRACT Activating mutations in ERBB2, the gene encoding the HER2 receptor tyrosine kinase (RTK), occur in ~3% of human tumors and correlate with poor prognosis in breast cancer. Since HER2 mutations usually occur in the absence of HER2 gene amplification, there are currently no approved therapies for this breast cancer subtype. A subset of patients with HER2-mutant breast cancers exhibit excellent clinical responses to anti-HER2 therapies such as the tyrosine kinase inhibitor (TKI) neratinib, suggesting that HER2 mutations are oncogenic drivers. However, the molecular mechanisms by which mutant HER2 promotes breast cancer progression are poorly understood and the response of the most common HER2 mutants to the multiple available anti-HER2 therapies has not been systematically investigated. Finally, durable clinical responses to HER2 TKIs are not the rule and are generally transient, suggesting mechanisms of drug resistance that remain to be discovered. Our objectives are to determine the mechanisms by which mutant HER2 promotes breast cancer oncogenesis and to identify the treatments that are most effective against HER2-mutant breast cancers. We hypothesize that 1) recurrent HER2 mutations generate gain-of-function activity and, as such, tumor dependence on aberrant HER2 signaling, which can be inhibited with targeted therapies; 2) HER2 mutations cooperate with co-occurring mutations in other ERBB RTKs to promote breast cancer growth; and 3) co-occurring genomic alterations will lead to intrinsic or acquired resistance to HER2 TKIs in HER2-mutant cancers. To test these hypotheses, we propose the following three aims: 1) To define mechanisms by which HER2 mutants promote breast oncogenesis and cancer progression; 2) To examine whether HER2 mutations cooperate with alterations in other ERBB RTKs; and 3) To identify mechanisms of resistance to HER2 TKIs in HER2-mutant breast cancers. We propose to integrate structural, biochemical, molecular and in vivo approaches to complete these aims. We will use computational modeling and phospho-protein arrays to determine the mechanisms by which HER2 mutations exert their tumorigenic properties, and will use inhibitors of HER2 signaling to block these effects in HER2-mutant cell lines and patient-derived xenografts (PDXs). We will model cooperation between observed co-occurring mutations in HER2/EGFR and HER2/HER3 in vitro and in vivo. We will employ a genome-wide genetic screen to identify genes that promote resistance to neratinib antiestrogens in HER2-mutant breast cancer cells. Finally, we will develop models of acquired resistance to neratinib using HER2-mutant PDXs and identify mechanisms of resistance by next-generation DNA and RNA sequencing, which will be confirmed in patient samples from the SUMMIT clinical trial. These studies will determine the best drug combinations to use in order to inhibit mutant HER2-driven cancer progression and will identify strategies to overcome resistance to HER2 mutation inhibitors. These studies have the potential to be rapidly translated to the clinic and significantly reduce mortality from HER2-mutant cancers.